Identification of deletion carriers in X-linked chronic granulomatous disease by real-time PCR.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency affecting the innate immune system. Even if functional tests address the diagnosis of CGD, the identification of a molecular defect is essential for counselling family members at risk for being CGD carriers and for prenatal diagnosis. The X-linked form occurs in 65% of CGD patients. It is due to mutations in the CYBB gene, up to 12% of which are caused by large deletions. CGD carriers are usually healthy, and molecular analysis is essential to reveal their carrier status. The aim of this study was to apply a gene dosage approach, using SYBR green quantitative real-time polymerase chain reaction (RT-PCR), to quantify the genomic copy number in carriers and noncarriers of gross deletions covering the region of the CYBB gene. We studied the expression of two different amplification products of the CYBB gene, and the results confirmed a highly reduced expression of the gene in the carrier samples. The results were confirmed by linkage analysis and fluorescence in situ hybridization. Quantitative real-time PCR is fast and simple to perform, and we propose it as a new routine diagnostic approach to detect CGD carriers of deletions covering the region spanning the CYBB gene.

Typing of ARMS2 and CFH in age-related macular degeneration: case-control study and assessment of frequency in the Italian population.

OBJECTIVES: To determine the effects of the polymorphisms CFH Tyr402His and ARMS2 del443ins54 on susceptibility to age-related macular degeneration (AMD) and to find the frequencies of these single-nucleotide polymorphisms in an Italian population that was not examined clinically. METHODS: A total of 286 control subjects (126 men and 160 women) and 159 white patients (73 men and 86 women) harboring exudative AMD in 1 eye were recruited. A third group of 182 DNA samples from blood donors of the same geographical areas were also typed to assess the frequency of CFH Tyr402His and ARMS2 del443ins54 polymorphisms in the general population. The data were analyzed statistically by a standard 2 x 2 table, Fisher exact tests, and odds ratios. RESULTS: The deletion-insertion at chromosome 10q26 (del443ins54) showed the strongest association with AMD in terms of both P value and odds ratio (P = 2.7 x 10(-15); odds ratio = 3.25), and a highly significant association was also confirmed for Tyr402His at the CFH locus (P = 9.9 x 10(-13); odds ratio = 2.86). We found no differences in allele and genotype association between classic and occult choroidal neovascularization. We also observed that 39% of the samples in the general Italian population were at least 5.4 times more likely than control subjects to develop AMD. CONCLUSIONS: To our knowledge, this is the first confirmation of the association of del443ins54 in Italian patients with AMD, and we also confirmed the association of Tyr402His with CFH. Genetic analysis of the general population suggested that analysis of the ARMS2 and CFH risk alleles alone may be helpful in differentiating high-risk individuals (odds ratio > 5.00) from low-risk individuals (odds ratio < 0.45). CLINICAL RELEVANCE: Individuals at high risk for developing AMD could be identified and selected for specific prevention programs. In this context, the development of prevention programs based on dietary antioxidants or on close monitoring of at-risk individuals should be considered or suggested.

A multiplex molecular assay for the detection of uniparental disomy for human chromosome 7.

Uniparental disomy (UPD) describes the inheritance of both homologues of a pair of chromosomes from only one parent. This condition is often caused by nondisjunction events during meiosis. UPD has been reported as a rare cause of the autosomal recessive disorder and aberrant expression of imprinted genes that are expressed from only one parental allele, either maternal or paternal. Maternal and/or paternal UPD for chromosome 7 is the most frequently observed UPD after UPD15. Here we developed and validated, for the first time, an effective, CE-based method for a rapid and economic detection based on two-fluorescent STR multiplexes.

Molecular characterization of a large cohort of patients with Chronic Granulomatous Disease and identification of novel CYBB mutations: an Italian multicenter study.

Chronic Granulomatous Disease (CGD) is a rare inherited disorder in which phagocytes fail to produce antimicrobial superoxide because NADPH oxidase activity is absent. In about 65% of the cases, the disease is due to mutations affecting the X-linked CYBB gene, encoding the gp91(phox) subunit of NADPH oxidase. We investigated 34 CGD male patients by DHPLC and direct sequencing. A mutation was found in the CYBB gene of 33 patients and 9 of these were novel: one non-sense mutation (c.1123 G>T), three missense mutations (c.58G>A; c.1076 G>C; c.1357 T>A), two splice site mutations (c.141+5G>T; c.142-1G>A), one duplication (c.42_45dupCATT), one deletion (c.184delT), and one rare deletion of two non-contiguous nucleotides (c.1287delT+c.1290delC). One patient had the most frequent GT homozygous deletion in exon2 of the NCF-1 gene encoding the p47(phox) subunit of NADPH oxidase. The carrier analysis was performed in 23 patients' mothers and 16 female relatives through molecular and FISH studies. No clear correlation between the severity of clinical symptoms and the type of mutation could be demonstrated. This study further supports the great heterogeneity of the disease and the notion that genetic analysis is a critical step in obtaining a definitive diagnosis for CGD.

A multiplex molecular assay for the detection of uniparental disomy for human chromosome 15.

Uniparental disomy (UPD) describes the inheritance of both homologues of a pair of chromosomes from only one parent. During the last two decades, the clinical impact of UPD and associated imprinting disorders, such as Prader-Willi syndrome (PWS) and Angelman syndrome (AS) increasingly have come to our attention. About 25% of PWS and 3%-5% of AS are a consequence of UPD with the resulting phenotype generated from the parent of origin of the disomic pair of chromosomes 15. Chromosome 15 UPD testing is relevant in various prenatal diagnostic conditions including apparent confined placental mosaicism, homologous and nonhomologous Robertsonian translocations involving chromosome 15 and 14, and as genomic biomarker for detecting chromosome origin. In this work we developed and validated a two fluorescent STRs multiplex assay for a rapid, economic and fully informative detection of UPD 15 by capillary electrophoresis.

Mannose-binding lectin polymorphisms and pulmonary outcome in premature neonates: a pilot study.

OBJECTIVE: Mannose-binding lectin (MBL2) is a collectin molecule able to activate the complement system and the subsequent inflammatory mechanisms. Several MBL2 genetic variants have been described, including the six variants studied in this report, which are those analyzed in most detail in the medical literature. DESIGN: The present study analyzes the prevalence of MBL2 gene variants in preterm newborns and associates individual genotypes with pulmonary outcome variables. All polymorphisms were analyzed by means of a commercially available reverse dot-blot kit. SETTING: Tertiary neonatal intensive care unit. PATIENTS AND PARTICIPANTS: Seventy-five consecutive preterm newborns. MEASUREMENTS AND RESULTS: Two variants were particularly analyzed: -550G > C and R52C. The first one is known to be associated with lower protein synthesis when included in specific haplotypes. The homozygous and heterozygous -550G > C mutations were significantly associated with protective effects regarding different lung outcome variables, including shorter duration of mechanical ventilation, hours of continuous positive airway pressure and lower number of hemotransfusions. In contrast, the heterozygous R52C mutation was associated with unfavorable outcome, including higher bronchopulmonary dysplasia prevalence. Multivariate logistic regression analysis showed that these associations were independent of gestational age and birth weight. In addition, four groups of patients were defined on the basis of haplotype combinations. Those known to be associated with low serum MBL2 levels were linked to a better outcome in terms of factors such as hours of mechanical ventilation, continuous positive airway pressure, number of hemotransfusions and bronchopulmonary disease development. CONCLUSIONS: The four haplotype combination groups may have a potential diagnostic use as opposite risk factors for lung disease of prematurity.

Mapping the future of common diseases: lessons from psoriasis.

Psoriasis (OMIM*177900) is a common, chronic, hyperproliferative inflammatory disorder of the skin affecting approximately 2% of Caucasians. Despite the prevalence of psoriasis in general population, significant differences in the incidence among Japanese, Eskimos, West Africans, north American blacks and American natives are well known. The cause for these variations are likely to be both genetic and environmental. Independent genomewide scans have suggested the involvement of a large number of chromosomal regions (loci), but so far only poor susceptibility genes have been suggested. We discuss genetic basis of the disease, results and interpretations of relevant studies, with particular regard to study design and future perspectives. Indeed to date, mapping genes which contribute to complex diseases is one of the major challenge in the post-genomic era.

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21.

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.

PSORS2 markers are not associated with psoriatic arthritis in the Italian population.

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy characterized by the association of arthritis and psoriasis (Ps). The precise etiology of PsA is unknown, but epidemiological studies supported the existence of a genetic component for the disease. Here we report an association study on a large PsA Italian cohort for DNA variants recently reported as associated alleles at PSORS2 (17q25) in Ps cohorts from the US. We focused on discovering a possible involvement of PSORS2 associated SNPs in pathogenesis of PsA. We selected two SNPs (rs7420, rs734232) within the proximal peak and two SNPs (rs869190 and rs1561946) within distal peak of PSORS2. Our results ruled out PSORS2 alleles as susceptibility factors in arthritis psoriatic patients of Italian origin and suggested that previous linkage signal reported for chromosome 17q25 should be independent on the presence of PsA.

The psoriasis genetics as a model of complex disease.

Psoriasis [OMIM*177900] is a common, chronic and papulosquamous inflammatory skin disease affecting approximately 2% of Caucasian. However, this disorder is rare among Japanese, Eskimos, West Africans and North American blacks and very uncommon in North American and South American natives. The causes for these variations are likely to be both genetic and environmental. Population-based studies and twin studies indicate that psoriasis is a heritable disease with a polygenic mode of inheritance with variable penetrance. Independent genome-wide scans have suggested the involvement of a large number of chromosomal regions (loci), and many candidate genes have been proposed. We discuss genetic approaches to the disease, results and interpretations of relevant studies, as well as future perspectives. Understanding the genetic basis of psoriasis will represent a major advance in our understanding of the disease and will reveal novel disease-specific biologic pathways.

Psoriatic arthritis and CARD15 gene polymorphisms: no evidence for association in the Italian population.

Psoriatic arthritis (PsA) has been defined as an inflammatory arthritis associated with psoriasis that may affect as many as 30% of psoriasis patients. Epidemiological study reported strong familial clustering of PsA although the precise etiology of PsA is poorly understood. Recently, a genomewide linkage scan in PsA revealed a LOD score of 2.17 on chromosome 16q and provided strong evidence for a paternal imprinting effect. That region surrounds a psoriasis susceptibility locus including the CARD15 gene which has convincingly been shown to confer susceptibility to Crohn's disease. The existence of a common susceptibility gene for psoriasis/PsA and Crohn disease was recently demonstrated by evidence of association of CARD15 polymorphisms with PsA. To confirm these results in an independent population, we analyzed a data set of 193 Italian PsA patients and 150 controls for CARD15 polymorphisms (R702W, G908R and leu1007finsC) previously demonstrated associated with PsA. Here we report no evidence for association in the examined population for CARD15 polymorphisms, suggesting that the positive association previously reported in a genetically isolated population was the result of a linkage disequilibrium due to a founder effect.